Deactivation of linking moieties in antibody-enzyme conjugates

ABSTRACT

In some embodiments, the present invention pertains to a method for conjugating a first compound to a second compound wherein the conjugation involves an electrophilic moiety. The method comprises reacting the first compound with the second compound to form a conjugate. The improvement in embodiments of the present invention comprises adding a nucleophilic reagent to the conjugate wherein the nucleophilic reagent forms a neutral product upon reaction with unreacted electrophilic moieties of the conjugate. In some embodiments, the nucleophilic reagent is substantially non-reactive with disulfide bonds in the event that the conjugate comprises disulfide bonds. The conjugate formed is doubly deactivated because the other moiety for linking to the electrophilic moiety is also deactivated.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the subject matter disclosed inprior Provisional Patent Application Ser. No. 60/605,106 filed Aug. 26,2004, the disclosure of which is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

In the fields of medicine and clinical chemistry, many studies anddeterminations of physiologically reactive species such as cells,proteins, enzymes, cofactors, nucleic acids, substrates, antigens,antibodies, and so forth are carried out using conjugates involvingspecific binding pair members or labels or the like. Various assaytechniques that involve the binding of specific binding pair members areknown. These assay techniques generally also involve a label used in thedetection part of the assay.

The use of enzyme labels for the determination of analytes inimmunoassays has shown substantial promise and numerous immunoassayshave been developed, which are dependent upon the accurate measurementof enzymatic activity from an assay medium.

Numerous enzyme immunoassay methods are known for the determination ofthe presence in a sample of an analyte. The assays include bothhomogeneous and heterogeneous assays. Some homogeneous approachescomprise determining the effect that the sample containing the analytehas on the binding between a conjugate of the analyte (ligand) and anenzyme and a receptor for the analyte. When the conjugate and thereceptor bind, a modulation of the enzymatic activity occurs. Thepresence of analyte in the sample can be determined from the effect thatthe analyte has on the modulation of the enzymatic activity whencompared to that obtained in the absence of analyte or in the presenceof known amounts of analyte. Heterogeneous assays include sandwichformat assays. The aforementioned assays are discussed in more detailbelow.

Polypeptides can be conjugated with enzymes using preactivated enzymessuch as, for example, enzymes preactivated with a maleimide or ahaloacetyl moiety. The polypeptides may comprise free sulfhydryl (—SH)groups or may be treated to introduce such groups. In the lattercircumstance, linking groups may be employed to link to the polypeptideto introduce sulfhydryl groups. Accordingly, the linking group may havean amine-reactive functionality in addition to the sulfhydryl groupwhere the amine reactive functionality reacts with free amine groups onthe polypeptide. Maleimide-activated enzyme is reacted with freesulfhydryl (—SH) groups present in, or introduced into, the polypeptideto form a stable thiol ether linkage.

In another approach, amino groups of a polypeptide are acylated with aheterobifunctional crosslinking agent such as N-succinimidyl4′-(p-maleimidophenyl) butyrate. This crosslinking agent has aN-hydroxysuccinimide group at one end of the molecule that reacts withamino groups. The maleimide moiety at the other end of theheterobifunctional agent reacts with free sulfhydryl groups of apolypeptide.

In the above reactions, it is standard practice to terminate theconjugation reaction with a quench reagent that deactivates one of thereactive groups that is used to form the conjugate, namely, freesulfhydryl groups remaining on the product after the conjugationreaction has taken place. The sulfhydryl groups are deactivated byadding a reagent reactive with the sulfhydryl groups such as, forexample, a sulfhydryl receptor, e.g., a maleimide, and so forth.

There remains a need for methods for producing ligand binder-polypeptideconjugates such as, for example, enzyme-polypeptide conjugates,polynucleotide-polypeptide conjugates, and so forth, that exhibit goodstability, activity, sensitivity, and the like.

SUMMARY

In some embodiments, the present invention pertains to a method forconjugating a first compound to a second compound wherein theconjugation involves an electrophilic moiety. The method comprisesreacting the first compound with the second compound to form aconjugate. The improvement in embodiments of the present inventioncomprises adding a nucleophilic reagent to the conjugate wherein thenucleophilic reagent forms a neutral product upon reaction withunreacted electrophilic moieties of the conjugate. In some embodiments,the nucleophilic reagent is substantially non-reactive with disulfidebonds in the event that the conjugate comprises disulfide bonds.

In some embodiments, the present invention is directed to a method forconjugating a first polypeptide to a second polypeptide wherein theconjugation involves a sulfhydryl receptor. The first polypeptide iscombined with the second polypeptide under conditions for the firstpolypeptide and the second polypeptide to react to form a conjugateinvolving the sulfhydryl receptor. Then, a sulfhydryl receptordeactivation reagent is added to the conjugate and the sulfhydrylreceptor deactivation reagent forms a neutral deactivated product uponreaction with the sulfhydryl receptor.

In some embodiments, the present invention is directed to a conjugatecomprising a first polypeptide and a second polypeptide linked togetherby sulfhydryl groups bound to sulfhydryl receptors. The sulfhydrylreceptors that are not bound to sulfhydryl groups are in the form of aneutral product of the reaction of a sulfhydryl receptor and asulfhydryl receptor deactivation reagent. In some embodiments, thesulfhydryl receptor deactivation reagent is substantially non-reactivewith disulfide linkages when the conjugate comprises disulfide bonds.

DESCRIPTION OF SPECIFIC EMBODIMENTS

The present invention provides a simple, inexpensive method forconjugation of macromolecules together to form conjugates that areuseful in assays such as, for example, immunoassays. Typically, asexplained above, the conjugation process involves two different reactivegroups or moieties. For the most part, the known techniques have focusedon deactivation of only one of the two residual reactive groups. Theconjugates of the invention have deactivated residual reactive groupsfor both of the reactive moieties that are used to form the conjugates.

As mentioned above, in some embodiments, the present invention pertainsto a method for conjugating a first compound to a second compoundwherein the conjugation involves at least one reactive moiety that is anelectrophilic moiety. The method comprises reacting the first compoundwith the second compound to form a conjugate. The improvement inembodiments of the present invention comprises adding a nucleophilicreagent to the conjugate subsequent to the conjugation reaction whereinthe nucleophilic reagent forms a neutral product upon reaction withunreacted or residual electrophilic moieties of the conjugate. In someembodiments, the nucleophilic reagent is substantially non-reactive withdisulfide bonds, which might be present in one or both of the twocompounds that are conjugated together. Furthermore, a reactive moietyused in the conjugation with the reactive electrophilic moiety may be,and in many embodiments is, deactivated also with a suitabledeactivation reagent thereby yielding a di-deactivated conjugate.

Conjugates

The first compound and the second compound are usually macromolecules,usually two different macromolecules including ligand receptors. Aligand is any organic compound for which a receptor naturally exists orcan be prepared and the ligand receptor is the receptor for such ligand.

The term “macromolecule” or “ligand receptor” includes biopolymers suchas, for example, polynucleotides, polypeptides, polysaccharides, and thelike, which generally have a molecular weight of at least about 5,000,more usually at least about 10,000. A biopolymer is a polymer of one ormore types of repeating units or monomers. Biopolymers are typicallyfound in biological systems. In the polypeptide, polysaccharide orpolynucleotide category, the molecules are generally from about 5,000 toabout 50,000,000 or more molecular weight, or from about 10,000 to about30,000,000 or more molecular weight, or about 20,000 to about 10,000,000molecular weight or more. Polypeptides are compounds or compositionsthat are poly(amino acids), that is, polymeric amino acids and aresometimes referred to as proteins. Polynucleotides are compounds orcompositions that are polymeric nucleotides or nucleic acid polymers.Polysaccharides are compounds or compositions that are polymericcarbohydrates.

The above terms also include analogs of the monomeric parts such as, forexample, those compounds composed of or containing amino acid analogs ornon-amino acid groups, or nucleotide analogs or non-nucleotide groups.This includes polynucleotides in which the conventional backbone hasbeen replaced with a non-naturally occurring or synthetic backbone, andnucleic acids (or synthetic or naturally occurring analogs) in which oneor more of the conventional bases has been replaced with a group(natural or synthetic) capable of participating in Watson-Crick typehydrogen bonding interactions.

A wide variety of proteins are included within the term “polypeptide.”Such proteins include proteins having particular biological functions,proteins related to specific microorganisms, particularly diseasecausing microorganisms, etc. Exemplary of such proteins areimmunoglobulins or antibodies, cytokines, enzymes, hormones, cancerantigens, nutritional markers, tissue specific antigens, avidin,streptavidin, folate binding proteins, hormone receptors, protein A,protein G, and so forth. Polynucleotides include single-stranded ordouble-stranded DNA, RNA, modified DNA, modified RNA, m-RNA, r-RNA,t-RNA, cDNA, DNA-RNA duplexes, etc.

Embodiments of the invention have application to the conjugation of manytypes of compounds where two or more reactive functionalities arereacted together to form the conjugate. A reactive functionality is agroup or moiety that is capable of reacting with another group ormoiety. Both groups or moieties, thus, are referred to as functionalgroups or moieties. The reactive functionality may be reactive by virtueof electrophilicity, nucleophilicity, basicity, acidity, free radicalactivity, photochemical activity, or the like. Such reactivefunctionalities include, for example, thiol or sulfhydryl, amine,carboxy, hydroxy, olefin, substituted olefin, epoxide, aldehyde, alkylhalide, and so forth.

A reactive functionality may be present on the compound to be conjugatednaturally or it may be introduced by chemical reaction, by introductionusing a heterobifunctional or homo-bifunctional reagent and so forth.Such chemical reactions to introduce a reactive functionality aredependent on the nature of the reactive functionality, the nature of themoiety to be reacted with, structural features of the macromoleculebeing modified, and the like. Such chemical reactions are apparent tothose skilled in the art.

A linking group may be employed to connect two or more substructures,namely, two or more compounds. The linking group may vary from a bond toa chain of from 1 to about 30 or more atoms, from about 1 to about 20atoms, from 1 to about 10 atoms, each independently selected from thegroup normally consisting of carbon, oxygen, sulfur, nitrogen, andphosphorous, usually carbon and oxygen. The number of heteroatoms in thelinking group may range from about 0 to about 8, from about 1 to about6, from about 2 to about 4. The number of atoms in the chain isdetermined by counting the number of atoms other than hydrogen or othermonovalent atoms along the shortest route between the substructuresbeing connected. The atoms of the linking group may be substituted withatoms other than hydrogen such as carbon, oxygen and so forth in theform, e.g., of alkyl, aryl, aralkyl, hydroxyl, alkoxy, aryloxy,aralkoxy, and the like. As a general rule, the length of a particularlinking group can be selected arbitrarily to provide for convenience ofsynthesis with the proviso that there be minimal interference caused bythe linking group with the ability of the compounds to be linkedtogether. The linking group may be aliphatic or aromatic.Functionalities present in the linking group may include esters,thioesters, amides, thioamides, ethers, ureas, thioureas, guanidines,azo groups, thioethers, carboxylate and so forth. Examples, by way ofillustration and not limitation, of various linking groups that may finduse in the present invention are found in U.S. Pat. No. 3,817,837,particularly at column 30, line 69, to column 36, line 10, whichdisclosure is incorporated herein by reference in its entirety. Theorder of reacting the linking group with the compounds may involvereacting one compound with the linking group followed by reaction withthe other compound. Simultaneous reaction is also possible but notpreferred in most instances. The order of reaction is determined by thenature of the linking group, the nature of the reactive functionalities,the nature of the compounds to be linked, the stability of the reactivecompounds and intermediates, and so forth.

Embodiments of the present invention have particular application to theconjugation of labels such as, for example, enzymes, phycobiliproteins,latex particles, radio-labeled proteins, fluorescent dye-labeledproteins, chromophore-labeled proteins, chemilumiflours, generic bindingproteins such as streptavidin, and the like with macromolecules orligand receptors such as, for example, antibodies, to prepare conjugatesuseful in assays such as, for example, immunoassays.Enzyme-macromolecule conjugates are typically used for the detection anddetermination of substances present in very low quantities, for example,picogram to nanogram quantities of substances in biological fluids, suchas urine and serum. A wide variety of enzymes may be used to form theconjugate, but the enzymes selected are often those enzymes that can bedetected with great sensitivity. In the preparation of enzymeconjugates, it is most desirable to produce enzyme conjugates such asenzyme antibody conjugates of high stability, high specificity and goodreproducibility. The nature of the enzyme selected for the conjugatedepends on the nature of the assay, the nature of the detection system,and the like.

The enzyme may be any enzyme that is suitable as a label for detectionin an assay. Such enzymes include, by way of illustration and notlimitation, beta-galactosidase, alkaline phosphatase,glucose-6-phosphate dehydrogenase (“G6PDH”), horseradish peroxidase,luciferases, kinases, oxidases, and so forth. Other enzymes andcoenzymes, which may be linked in place of an enzyme, are disclosed inLitman, et al., U.S. Pat. No. 4,275,149, columns 19-28, and Boguslaski,et al., U.S. Pat. No. 4,318,980, columns 10-14; suitable fluorescers andchemiluminescers are disclosed in Litman, et al., U.S. Pat. No.4,275,149, at columns 30 and 31; which are incorporated herein byreference.

The antibody as ligand receptor can be monoclonal or polyclonal and canbe prepared by techniques that are well known in the art such asimmunization of a host and collection of sera (polyclonal) or bypreparing continuous hybrid cell lines and collecting the secretedprotein (monoclonal), or by cloning and expressing nucleotide sequencesor mutagenized versions thereof coding at least for the amino acidsequences required for specific binding of natural antibodies.Antibodies may include a complete immunoglobulin or fragment thereof,which immunoglobulins include the various classes and isotypes, such asIgA, IgD, IgE, IgG1, IgG2a, IgG2b and IgG3, IgM, etc. Fragments thereofmay include Fab, Fv and F(ab′)₂, Fab′, and the like. In addition,aggregates, polymers, and conjugates of immunoglobulins or theirfragments can be used where appropriate so long as binding affinity fora particular molecule is maintained.

One of the linking moieties is usually an electrophilic moiety such as,for example, a moiety comprising a sulfhydryl receptor, an aldehyde, anactive ester, and the like. The electrophilic moiety may be part of amono-functional reagent where the agent comprises only one linkingmoiety. On the other hand, the electrophilic moiety may be part of abi-functional reagent, a tri-functional reagent, and so forth. Suchreagents comprise one or more functionalities for linking other than theelectrophilic moiety.

Examples of sulfhydryl receptors are carbonyl-substituted olefins,sulfone-substituted olefins, carbonyl substituted, halogen substitutedcarbon atoms, pyridyl disulfide groups, epoxides, and so forth. Thecarbonyl substituted olefin is generally an olefin (carbon-carbon doublebond) that may be substituted with one, two, or more carbonyl groups.The carbonyl groups include, for example, carboxyl, carboxamide,carboximide, carboxylic ester, anhydride, aldehyde, ketone, and thelike. One example of a carbonyl substituted olefin is a maleimide ormaleimidyl moiety, which includes, by way of illustration and notlimitation, N-(4-carboxycyclohexylmethyl)maleimide,N-(m-benzoyl)maleimide, m-maleimido-benzoyl-N-hydroxysulfosuccinimideester, succinimidyl maleimidylacetate, and succinimidyl6-maleimidylhexanoate, N-succinimidyl 4′-(p-maleimidophenyl) butyrate,meta-maleimidobenzoyl N-hydroxysuccinimide ester, N-hydroxysuccinimideester of N-(m-benzoyl)maleimide,N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), and the like.

Examples of halogen substituted, carbonyl substituted carbon atomsinclude iodoacetic acid, iodoacetic anhydride, N-hydroxysuccinimideester of iodoacetic acid, other active esters of iodoacetic acid, andthe like. The sulfone-substituted olefins include compounds wherein thesulfone bond electrons are conjugated with the electrons of the olefinbond, i.e., the olefin bond is conjugated with the sulfone moiety.

Groups reactive with the sulfhydryl receptor to form the enzyme antibodyconjugates include, for example, sulfhydryl, e.g., thiol or mercaptan,hydrazine derivative, hydrazide, amino-oxy ether, and the like.Sulfhydryl groups may be present naturally in the molecule to beconjugated or they may be introduced into the molecule such as bychemical reaction and the like. Methods for introducing sulfhydrylgroups may be found in “Enzyme Immunoassay,” by Eiji Ishikawa,(Lippincott Williams & Wilkins, July 1981.

The reaction conditions employed in the formation of the conjugatebetween an enzyme and an antibody where one of the molecules comprises asulfhydryl group (either naturally or introduced) and the other of themolecules comprises a sulfhydryl receptor (either naturally orintroduced) usually involves bringing a reaction mixture comprising theabove to a pH in the range of about 5-10, more usually in the range ofabout 6-9. Various buffers may be used such as phosphate, carbonate,Tris, and the like. An aqueous solvent is normally used and up to about40 weight % of an oxyethylene alcohol or ether having from 1 to 3oxyethylene units may be present. The temperatures normally range fromabout −5° C. to about 40° C., usually from about 0° C. to about 25° C.Conditions for the reaction are usually determined by the nature of theantibody and/or the enzyme, the nature of the linking moieties and soforth.

Deactivation of Linking Moieties in Conjugates

As mentioned above, in the above reactions, it is standard practice toterminate the conjugation reaction with a quench reagent thatdeactivates one of the reactive groups that is used to form theconjugate, namely, free sulfhydryl groups remaining on the product afterthe conjugation reaction has taken place. The free sulfhydryl groups aredeactivated by adding a reagent reactive with the sulfhydryl groups suchas, for example, a sulfhydryl receptor, e.g., a maleimide (N-ethylmaleimide and other maleimides discussed above), reagents comprisingsulfhydryl receptors other than maleimide as discussed above, and soforth.

We have found that, in addition to deactivation of nucleophilic moietiessuch as remaining sulfhydryl groups, it is advantageous to deactivatefree electrophilic moieties such as sulfhydryl receptors remaining onthe product after conjugation thereby yielding a di-deactivatedconjugate. This is counter-intuitive because one might reasonablyconclude that deactivating one of the reactive groups would therebysubstantially reduce or eliminate any reactions that might occur betweenthe free groups. Furthermore, an enhancement in specific activity of theconjugates is realized as explained more fully below.

In some embodiments of the invention, a nucleophilic reagent is added tothe conjugate subsequent to formation wherein the nucleophilic reagentforms a neutral product upon reaction with unreacted electrophilicmoieties of the conjugate. The term “neutral product” means that theresulting deactivated moiety is free of a positive or negative chargesuch as might be found with a carboxylic acid functionality, aminefunctionality, and so forth, resulting from the reaction of thenucleophilic moiety with the electrophilic moiety of the conjugate. Insome embodiments the neutral product results from the formation of aring structure or cyclized product upon reaction of the deactivationreagent with the electrophilic moiety. Consequently, in some embodimentsthe nucleophilic reagent is one that results in the formation of astable ring structure or cyclized product upon reaction of thenucleophilic reagent with free electrophilic moieties remaining in theconjugate. In some embodiments, the deactivation reagent or nucleophilicreagent is substantially non-reactive with disulfide bonds in the eventthat the conjugate comprises disulfide bonds. By “substantiallynon-reactive” is meant that reaction with disulfide bonds does not occurto an extent that the activity of the moiety comprising the disulfidebonds is changed to any appreciable extent such that its effectivenessis compromised.

The deactivated product should exhibit good activity and stability. Adesirable goal for stability is that there must be less than a 5% and insome embodiments less than a 4%, less than a 3%, less than a 2% changein response over one month for product stored at 4° C. Response isdefined as recovered analyte, which is generally proportional to theresponse of the assay. For example, if the assay response is a change inoptical density due to enzymatic activity with a chromogenic substrate,the response seen in the assay will be measured in absorbance units. Inthe above example, the assay response in absorbance units should changeby less than 5% over one month in order to obtain the desired goal forstability.

Deactivation reagents for deactivating the electrophilic moiety, forexample, a sulfhydryl receptor, include compounds of the followingformula:R²HN—(R₁)_(n)—NHR²wherein n is 0 or 1, R¹ is alkylene or substituted alkylene and R² isindependently H, alkyl or substituted alkyl. When n is 0, there is adirect bond between the nitrogen atoms. Alkylene means a branched orunbranched saturated divalent hydrocarbon radical. In some embodimentsalkylene includes, for example, alkylene of 1 to about 5 carbon atoms,alkylene of 1 to about 4 carbon atoms, alkylene of 1 to about 3 carbonatoms, alkylene of 1 to 2 carbon atoms, alkylene of about 2 to about 5carbon atoms, alkylene of about 2 to about 4 carbon atoms, alkylene ofabout 2 to about 3 carbon atoms, alkylene of about 3 to about 5 carbonatoms, alkylene of about 3 to about 4 carbon atoms, alkylene of about 4to about 5 carbon atoms. Particular examples include methylene,ethylene, propylene, 2-methylpropylene, 1,2-dimethylpropylene, butylene,pentylene, and the like.

Alkyl means a monovalent branched or unbranched radical derived from analiphatic hydrocarbon by removal of one H atom; includes both loweralkyl and upper alkyl.

Lower alkyl means alkyl containing from 1 to 5 carbon atoms, from 1 toabout 4 carbon atoms, from 1 to about 3 carbon atoms, from 1 to 2 carbonatoms, from about 2 to about 5 carbon atoms, from about 2 to about 4carbon atoms, from about 2 to about 3 carbon atoms, from about 3 toabout 5 carbon atoms, from about 3 to about 4 carbon atoms, from about 4to about 5 carbon atoms, such as, e.g., methyl, ethyl, propyl, butyl,isopropyl, isobutyl, pentyl, isopentyl, etc.

Upper alkyl means alkyl containing more than about 6 carbon atoms,usually about 6 to about 30 carbon atoms, about 10 to about 20 carbonatoms, about 6 to about 25 carbon atoms, about 6 to about 20 carbonatoms, about 10 to about 15 carbon atoms, such as, e.g., hexyl, heptyl,octyl, etc.

Substituted means that a hydrogen atom of a molecule has been replacedby another atom, which may be a single atom or part of a group of atomsforming a functionality as described above. Such substituents include byway of illustration and not limitation alkyl, and so forth.

In some embodiments the above deactivation reagents are desirablebecause they are not reactive with disulfide bonds where the conjugatecomprises a component that contains disulfide bonds such as, forexample, where the conjugate comprises an antibody. In some embodimentsthe deactivation agent for electrophilic moieties includes, for example,hydrazine, alkylene diamines of 1 to 5 carbon atoms such as, forexample, methylene diamine (diaminomethane), ethylene diamine(1,2-diaminoethane), propylene diamine (1,3-diaminopropane), and soforth, substituted hydrazines such as, for example, alkyl (lower alkyland upper alkyl) substituted hydrazine, substituted diamines such as,for example, alkyl (lower alkyl and upper alkyl) substituted diamines,and the like.

Mercaptans are also useful as deactivation reagents for electrophilicmoieties in some embodiments of the invention where a member orcomponent of the conjugate does not comprise disulfide bonds. Themercaptans are desirably free of amino groups. For embodiments where amember of the conjugate contains disulfides, it is desirable to employmercaptans that do not reduce the disulfides. Examples of suitablemercaptans include, by way of illustration and not limitation,thiophenol, and other thiols that have less reducing potential than thethiols generated when biological disulfides are cleaved, and so forth.Thiols that are strong reducing agents and, therefore, could cleave thedisulfides that maintain the tertiary structure of polypeptides of theconjugate are undesirable because they destroy the activity of thesepolypeptides.

The amount of the nucleophilic deactivation reagent to be added todeactivate residual sulfhydryl receptors of the conjugate depends on thenature of the electrophilic moiety and the nucleophilic deactivationreagent, the nature of the components of the conjugate such as, forexample, the number of expected free electrophilic moieties in theconjugate product, the rate of reaction, and so forth. Usually, theamount of the nucleophilic deactivation reagent added is in excess ofthe expected amount needed so that substantially all of the freeelectrophilic moieties are deactivated. The amount is usually determinedempirically and is the amount sufficient to deactivate the freeelectrophilic moieties of the conjugate to obtain a conjugate exhibitingenhanced activity.

The reaction conditions employed to deactivate the residual sulfhydrylreceptors are dependent on the nature of the deactivation reagent, thenature of the sulfhydryl receptor, the chemical reactivity of thecomponents of the conjugate, and so forth. For the most, particularreaction conditions will be evident to those skilled in the art withoutundue experimentation based on the disclosure herein and informationavailable in the art. Usually, an aqueous medium is employed, which maycomprise one or more organic solvents such as alcohols, ethers, amides,sulfoxides, sulfones, esters, and the like in a percentage range ofabout 1% to about 50% or more. The temperature employed for thedeactivation reaction may be, for example, about 0° C. to about 100° C.,about 5° C. to about 50° C., about 10° C. to about 40° C., about 15° C.to about 30° C. The deactivation reaction is carried out for a timeperiod sufficient for reactions to occur. Usually, the deactivation iscarried out, for example, for a period of about 1 hour to about 48hours.

Use of Deactivated Conjuqates

The di-deactivated conjugates of the present invention can be utilizedin many known assays for analytes. The assay methods usually involve asample suspected of containing an analyte, which is combined in an assaymedium with reagents for carrying out the assay. Such reagents caninclude a binding partner for the analyte such as, e.g., an antibody forthe analyte, analyte analogs, solid surfaces to which one of the abovereagents is bound, binding partners for sbp members, and so forth. Oneor more of the reagents can be labeled with a label such as, e.g., anenzyme. The reagents are chosen such that a signal is obtained from alabel in relation to the presence or amount of analyte in the sample.The assay can be performed either without separation (homogeneous) orwith separation (heterogeneous) of any of the assay compounds orproducts.

Homogeneous immunoassays are exemplified by the EMIT® assay products(Syva Company, San Jose, Calif.) disclosed in Rubenstein, et al., U.S.Pat. No. 3,817,837, column 3, line 6 to column 6, line 64; enzymechanneling techniques such as those disclosed in Maggio, et al., U.S.Pat. No. 4,233,402, column 6, line 25 to column 9, line 63; and otherenzyme immunoassays such as the enzyme linked immunosorbant assay(“ELISA”) are discussed in Maggio, E. T., infra. The above disclosuresare all incorporated herein by reference.

Heterogeneous assays usually involve one or more separation steps andcan be competitive or non-competitive. A variety of competitive andnon-competitive heterogeneous assay formats are disclosed in Davalian,et al., U.S. Pat. No. 5,089,390, column 14, line 25 to column 15, line9, incorporated herein by reference. In a typical competitiveheterogeneous assay a support having an antibody for analyte boundthereto is contacted with a medium containing the sample and analyteanalog conjugated to a detectable label such as an enzyme conjugate.Analyte in the sample competes with the conjugate for binding to theantibody. After separating the support and the medium, the labelactivity of the support or the medium is determined by conventionaltechniques and is related to the amount of analyte in the sample.

A typical non-competitive sandwich assay is an assay disclosed in David,et al., U.S. Pat. No. 4,486,530, column 8, line 6 to column 15, line 63,incorporated herein by reference. In this method, an immune sandwichcomplex is formed in an assay medium. The complex comprises the analyte,a first antibody (monoclonal or polyclonal) that binds to the analyteand a second antibody that binds to the analyte or a complex of theanalyte and the first antibody. Subsequently, the immune sandwichcomplex is detected and is related to the amount of analyte in thesample. The immune sandwich complex is detected by virtue of thepresence in the complex of a label wherein either or both the firstantibody and the second antibody contain labels or substituents capableof combining with labels.

Sandwich assays find use for the most part in the detection of antigenand receptor analytes. In the assay two antibodies specific for theanalyte bind the analyte and, thus, the assay is also referred to as thetwo-site immunometric assay. In one approach a first incubation ofunlabeled antibody coupled to a support, otherwise known as theinsolubilized antibody, is contacted with a medium containing a samplesuspected of containing the analyte. After a wash and separation step,the support is contacted with a medium containing the second antibody,which generally contains a label, for a second incubation period. Thesupport is again washed and separated from the medium and either themedium or the support is examined for the presence of label. Thepresence and amount of label is related to the presence or amount of theanalyte. For a more detailed discussion of this approach see U.S. Pat.Nos. Re 29,169 and 4,474,878, the relevant disclosures of which areincorporated herein by reference.

In a variation of the above sandwich assay, the sample in a suitablemedium is contacted with labeled antibody for the analyte and incubatedfor a period of time. Then, the medium is contacted with a support towhich is bound a second antibody for the analyte. After an incubationperiod, the support is separated from the medium and washed to removeunbound reagents. The support or the medium is examined for the presenceof the label, which is related to the presence or amount of analyte. Fora more detailed discussion of this approach see U.S. Pat. No. 4,098,876,the relevant disclosure of which is incorporated herein by reference.

In another variation of the above, the sample, the first antibody boundto a support and the labeled antibody are combined in a medium andincubated in a single incubation step. Separation, wash steps andexamination for label are as described above. For a more detaileddiscussion of this approach see U.S. Pat. No. 4,244,940, the relevantdisclosure of which is incorporated herein by reference.

Detection of antibodies is a useful tool in the diagnosis of infectiousdiseases. Detection of autoantibodies is also useful in the diagnosis ofautoimmune disease. Some of the above assay methods can be used todetect numerous antibodies such as antibodies to human immunodeficiencyvirus (“HIV”), rubella or herpes and autoantibodies such asautoantibodies to insulin, to glutamic acid decarboxylase (“GAD”), boththe 65 kd and the 67 kd isoforms but more particularly, GAD₆₅; and toother islet cell antigens.

The following provides a description of some of the terms used abovewith respect to the described assays.

Member of a specific binding pair (“sbp member”) means one of twodifferent molecules, having an area on the surface or in a cavity, whichspecifically binds to and is thereby defined as complementary with aparticular spatial and polar organization of the other molecule. Themembers of the specific binding pair are referred to as ligand andreceptor (antiligand). These will usually be members of an immunologicalpair such as antigen-antibody, although other specific binding pairssuch as biotin-avidin, hormones-hormone receptors, nucleic acidduplexes, IgG-protein A, polynucleotide pairs such as DNA-DNA, DNA-RNA,and the like are not immunological pairs but are included in thedefinition of sbp member for the purpose of describing this invention.

Analyte means the compound or composition to be detected. The analytecan be comprised of a member of a specific binding pair (sbp) and may bea ligand, which is usually monovalent, usually haptenic, or a ligandreceptor, which is usually polyvalent, and is a single compound orplurality of compounds that share at least one common epitopic ordeterminant site.

The monovalent ligand analytes will generally be from about 100 to 2,000molecular weight, more usually, from 125 to 1,000 molecular weight. Theanalytes include drugs, metabolites, pesticides, pollutants, and thelike. Representative analytes, by way of example and not limitation,include (i) alkaloids such as morphine alkaloids, which includemorphine, codeine, heroin, dextromethorphan, their derivatives andmetabolites; cocaine alkaloids, which include cocaine and benzylecgonine, their derivatives and metabolites; ergot alkaloids, whichinclude the diethylamide of lysergic acid; steroid alkaloids; iminazoylalkaloids; quinazoline alkaloids; isoquinoline alkaloids; quinolinealkaloids, which include quinine and quinidine; diterpene alkaloids,their derivatives and metabolites; (ii) steroids, which include theestrogens, androgens, andreocortical steroids, bile acids, cardiotonicglycosides and aglycones, which includes digoxin and digoxigenin,saponins and sapogenins, their derivatives and metabolites; steroidmimetic substances, such as diethylstilbestrol; (iii) lactams havingfrom 5 to 6 annular members, which include the barbiturates, e.g.,Phenobarbital and secobarbital, diphenylhydantoin, primidone,ethosuximide, and their metabolites; (iv) aminoalkylbenzenes, with alkylof from 2 to 3 carbon atoms, which include the amphetamines;catecholamines, which include ephedrine, L-dopa, epinephrine; narceine;papaverine; and metabolites of the above; (v) benzheterocyclics whichinclude oxazepam, chlorpromazine, tegretol, their derivatives andmetabolites, the heterocyclic rings being azepines, diazepines andphenothiazines; (vi) purines, which includes theophylline, caffeine,their metabolites and derivatives; (vii) drugs derived from marijuana,which include cannabinol and tetrahydrocannabinol; (viii) hormones suchas thyroxine, cortisol, triiodothyronine, testosterone, estradiol,estrone, progesterone, polypeptides such as angiotensin, LHRH, andimmunosuppressants such as cyclosporin, FK506, mycophenolic acid (MPA),and so forth; (ix) vitamins such as A, B, e.g. B12, C, D, E and K, folicacid, thiamine; (x) prostaglandins, which differ by the degree and sitesof hydroxylation and unsaturation; (xi) tricyclic antidepressants, whichinclude imipramine, dismethylimipramine, amitriptyline, nortriptyline,protriptyline, trimipramine, chlomipramine, doxepine, anddesmethyldoxepin; (xii) anti-neoplastics, which include methotrexate;(xiii) antibiotics, which include penicillin, chloromycetin,actinomycetin, tetracycline, terramycin, the metabolites andderivatives; (xiv) nucleosides and nucleotides, which include ATP, NAD,FMN, adenosine, guanosine, thymidine, and cytidine with theirappropriate sugar and phosphate substituents; (xv) miscellaneousindividual drugs which include methadone, meprobamate, serotonin,meperidine, lidocaine, procainamide, acetylprocainamide, propranolol,griseofulvin, valproic acid, butyrophenones, antihistamines,chloramphenicol, anticholinergic drugs, such as atropine, theirmetabolites and derivatives; (xvi) metabolites related to diseasedstates include spermine, galactose, phenylpyruvic acid, and porphyrinType 1; (xvii) aminoglycosides, such as gentamicin, kanamicin,tobramycin, and amikacin; and (xviii) pesticides such as polyhalogenatedbiphenyls, phosphate esters, thiophosphates, carbamates, polyhalogenatedsulfenamides, their metabolites and derivatives.

Polyvalent analytes are normally poly(amino acids), i.e., polypeptidesand proteins, polysaccharides, nucleic acids, and combinations thereof.Such combinations include components of bacteria, viruses, chromosomes,genes, mitochondria, nuclei, cell membranes and the like. For the mostpart, the polyvalent ligand analytes will have a molecular weight of atleast about 5,000, more usually at least about 10,000. In the poly(aminoacid) category, the poly(amino acids) of interest will generally be fromabout 5,000 to 5,000,000 molecular weight, more usually from about20,000 to 1,000,000 molecular weight; among the hormones of interest,the molecular weights will usually range from about 5,000 to 60,000molecular weight.

A wide variety of proteins may be considered as to the family ofproteins having similar structural features, proteins having particularbiological functions, proteins related to specific microorganisms,particularly disease causing microorganisms, etc. Such proteins include,for example, immunoglobulins, cytokines, enzymes, hormones, cancerantigens, nutritional markers, tissue specific antigens, etc. Suchproteins include, by way of illustration and not limitation, protamines,histones, albumins, globulins, scleroproteins, phosphoproteins,mucoproteins, chromoproteins, lipoproteins, nucleoproteins,glycoproteins, T-cell receptors, proteoglycans, HLA, unclassifiedproteins, e.g., somatotropin, prolactin, insulin, pepsin, proteins foundin human plasma, blood clotting factors, protein hormones such as, e.g.,follicle-stimulating hormone, luteinizing hormone, luteotropin,prolactin, chorionic gonadotropin, tissue hormones, cytokines, cancerantigens such as, e.g., PSA, CEA, a-fetoprotein, acid phosphatase,CA19.9 and CA125, tissue specific antigens, such as, e.g., alkalinephosphatase, myoglobin, CPK-MB and calcitonin, and peptide hormones.Other polymeric materials of interest are mucopolysaccharides andpolysaccharides.

For receptor analytes, the molecular weights will generally range from10,000 to 2×10⁸, more usually from 10,000 to 10⁶. For immunoglobulins,IgA, IgG, IgE and IgM, the molecular weights will generally vary fromabout 160,000 to about 10 ⁶. Enzymes will normally range from about10,000 to 1,000,000 in molecular weight. Natural receptors vary widely,generally being at least about 25,000 molecular weight and may be 10 ⁶or higher molecular weight, including such materials as avidin, DNA,RNA, thyroxine binding globulin, thyroxine binding prealbumin,transcortin, etc.

The term analyte further includes oligonucleotide and polynucleotideanalytes such as m-RNA, r-RNA, t-RNA, DNA, DNA-RNA duplexes, etc.

The analyte may be a molecule found directly in a sample such asbiological tissue, including body fluids, from a host. The sample can beexamined directly or may be pretreated to render the analyte morereadily detectable by removing unwanted materials. The sample may bepretreated to separate or lyse cells; precipitate, hydrolyse or denatureproteins; hydrolyze lipids; solubilize the analyte; or the like. Suchpretreatment may include, without limitation: centrifugation; treatmentof the sample with an organic solvent, for example, an alcohol, such asmethanol; and treatment with detergents. The sample can be prepared inany convenient medium, which does not interfere with an assay. Anaqueous medium is preferred.

The analyte of interest may be determined by detecting an agentprobative of the analyte of interest such as a specific binding pairmember complementary to the analyte of interest, whose presence will bedetected only when the analyte of interest is present in a sample. Thus,the agent probative of the analyte becomes the analyte that is detectedin an assay.

The biological tissue includes excised tissue from an organ or otherbody part of a host and body fluids, for example, urine, whole blood,plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid,tears, mucus, and the like. Preferably, the sample is plasma or serum.

Hapten means a compound capable of binding specifically to correspondingantibodies, but does not itself act as an immunogen (or antigen) forpreparation of the antibodies. Antibodies which recognize a hapten canbe prepared against compounds comprised of the hapten linked to animmunogenic (or antigenic) carrier. Haptens are a subset of ligands.

Ligand analog means a modified ligand, an organic radical or analyteanalog, usually of a molecular weight greater than 100, which cancompete with the analogous ligand for a receptor, the modificationproviding means to join a ligand analog to another molecule. The ligandanalog will usually differ from the ligand by more than replacement of ahydrogen with a bond which links the ligand analog to a hub or label,but need not. The ligand analog can bind to the receptor in a mannersimilar to the ligand. The analog could be, for example, an antibodydirected against the idiotype of an antibody to the ligand.

Specific binding means the specific recognition of one of two differentmolecules for the other compared to substantially less recognition ofother molecules. Generally, the molecules have areas on their surfacesor in cavities, also referred to as “binding sites,” giving rise tospecific recognition between the two molecules. Exemplary of specificbinding are antibody-antigen interactions, enzyme-substrateinteractions, polynucleotide interactions, and so forth.

Non-specific binding means non-covalent binding between molecules thatis relatively independent of specific surface structures. Non-specificbinding may result from several factors including hydrophobicinteractions between molecules.

Support or surface means a solid phase, typically a support or surface,which is a porous or non-porous water insoluble material that can haveany one of a number of shapes, such as strip, rod, plate, well, particleor bead. A wide variety of suitable supports are disclosed in Ullman, etal., U.S. Pat. No. 5,185,243, columns 10-11, Kurn, et al., U.S. Pat. No.4,868,104, column 6, lines 21-42, and Milburn, et al., U.S. Pat. No.4,959,303, column 6, lines 14-31, which are incorporated herein byreference.

The surface can be hydrophilic or capable of being rendered hydrophilicand includes inorganic powders such as silica, magnesium sulfate, andalumina; natural polymeric materials, particularly cellulosic materialsand materials derived from cellulose, such as fiber containing papers,e.g., filter paper, chromatographic paper, etc.; synthetic or modifiednaturally occurring polymers, such as nitrocellulose, cellulose acetate,poly (vinyl chloride), polyacrylamide, cross linked dextran, agarose,polyacrylate, polyethylene, polypropylene, poly(4-methylbutene),polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon,poly(vinyl butyrate), etc.; either used by themselves or in conjunctionwith other materials; glass available as Bioglass, ceramics, metals, andthe like. Natural or synthetic assemblies such as liposomes,phospholipid vesicles, and cells can also be employed.

Binding of sbp members to a support or surface may be accomplished bywell-known techniques, commonly available in the literature. See, forexample, “Immobilized Enzymes,” Ichiro Chibata, Halsted Press, New York(1978) and Cuatrecasas, J. Biol. Chem. , 245:3059 (1970).

Signal producing system (“sps”) means one or more components, at leastone component being a detectable label such as, e.g., an enzyme, whichgenerate a detectable signal that relates to the amount of bound and/orunbound label, i.e. the amount of label bound or not bound to thecompound being detected. Thus, the signal is detected and/or measured bydetecting enzyme activity, luminescence, radioactivity, or the like, asthe case may be.

There are numerous methods by which the label can produce a signaldetectable by external means, desirably by visual examination, forexample, by electromagnetic radiation, heat, and chemical reagents. Thelabel or other sps members can also be bound to an sbp member, anothermolecule or to a support.

In some embodiments, the label may need other components to produce asignal, and the signal producing system would then include all thecomponents required to produce a measurable signal, which may includesubstrates, coenzymes, enhancers, additional enzymes, substances thatreact with enzymic products, catalysts, activators, cofactors,inhibitors, scavengers, metal ions, dyes, fluorophores, radionucleides,and a specific binding substance required for binding of signalgenerating substances. A detailed discussion of suitable signalproducing systems can be found in Ullman, et al., U.S. Pat. No.5,185,243, columns 11-13, incorporated herein by reference.

The label can directly produce a signal and, therefore, additionalcomponents are not required to produce a signal. Phycobiliproteins, forexample, are able to absorb light, where the light absorption transfersenergy to these molecules and elevates them to an excited energy state.This absorbed energy is then dissipated by emission of light at a secondwavelength.

The label and/or other sps member may be bound to an sbp member or to asupport utilizing well-known procedures. Alternatively, the label can bebound covalently to an sbp member such as, for example, an antibody; areceptor for an antibody, a receptor that is capable of binding to asmall molecule conjugated to an antibody, or a ligand analog. Bonding ofthe label to the sbp member may be accomplished by chemical reactionsthat result in replacing a hydrogen atom of the label with a bond to thesbp member or may include a linking group between the label and the sbpmember. Other sps members may also be bound covalently to sbp members.See, for example, Rubenstein, et al., U.S. Pat. No. 3,817,837,incorporated herein by reference.

Assay means a method for the determination of the presence or amount ofan analyte.

Measuring the amount of an analyte means quantitative, semiquantitative,and qualitative methods as well as all other methods for determining ananalyte. For example, a method which merely detects the presence orabsence of an analyte in a sample suspected of containing the analyte isconsidered to be included within the scope of the present invention. Theterms “detecting” and “determining,” as well as other common synonymsfor measuring, are contemplated within the scope of the presentinvention.

Ancillary Materials means various materials frequently employed in theassay in accordance with the present invention. For example, bufferswill normally be present in the assay medium, as well as stabilizers forthe assay medium and the assay components. Frequently, in addition tothese additives, proteins may be included, such as albumins; organicsolvents such as formamide; quaternary ammonium salts; polyanions suchas dextran sulfate; surfactants, particularly non-ionic surfactants;binding enhancers, e.g., polyalkylene glycols; or the like.

Wholly or partially sequentially refers to the combining of variousagents other than concomitantly (simultaneously). One or more of thevarious agents may be combined with one or more of the remaining agentsto form a subcombination.

The invention is demonstrated further by the following illustrativeexamples.

EXAMPLES

Parts and percentages herein are by weight unless otherwise indicated.Temperatures are in degrees Centigrade (° C.).

Materials:

The hCG (human chorionic gonadotropin) and CKMB (creatinine kinase, MBsubtype) antibody Fab′2 fragments were prepared by a standard pepsinenzyme digestion process of mouse monoclonal antibodies that were grownin vitro. β-galactosidase was purchased from Roche DiagnosticsCorporation (Roche Applied Science, Indianapolis Ind.). N-ethylmaleimide(NEM) andsulfosuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC)linker were purchased from EMD Biosciences (La Jolla Calif.). All otherchemicals were purchased from the Sigma-Aldrich Company (St. Louis Mo.).

Testing was done using the DIMENSION® RxL analyzer, available from DadeBehring Inc., Deerfield, Ill.

Example 1

Part A

A solution of 250 mg of anti-hCG Fab′2 antibody was prepared at aconcentration of 8-10 mg/mL in 10 mM pH 7.0 phosphate buffer containing300 mM NaCl. To this solution, 2.507 mL of a 10 mg/mL SMCC solution inDMF (dimethylformamide) was added dropwise with stirring. The mixturewas then incubated for 30 minutes at 25° C., and then purified bypassage through a SEPHADEX G-25® size exclusion column. The elutionbuffer was the same as the reaction buffer. The concentration of productwas then determined using the OD280 (assuming the extinction coefficientis 1.4), and the concentration was adjusted to 2 mg/mL by addition ofadditional reaction buffer.

Part B.

A solution of 1350 mg of β-galactosidase in 675 mL of 10 mM pH 7.0phosphate buffer containing 300 mM NaCl was prepared, and brought to 25°C. with stirring. When the solution had reached this temperature thesolution of activated antibody from Part A above was added. The mixturewas incubated at 25° C. for 2 hours and 20 minutes, and then thereaction was stopped by adding 16 mL of a 100 mM stock solution ofN-ethylmaleimide (NEM) in water.

Part C.

15 minutes after addition of the NEM solution, 2 mL aliquots wereremoved from the product mixture. Different nucleophilic reagents,including sodium azide, sodium cyanide, thiophenol, hydrazine, andmercaptoethylamine (MEA) were added to the aliquots to giveconcentrations of 5, 25, and 125 mM nucleophiles. These mixtures wereheld for 24 hours at room temperature, and then refrigerated over nightat 4° C., at which time they were filtered through 0.2 micron filtersand then diluted 67-fold into the conjugate diluent used for theDIMENSION® RxL commercially available hCG conjugate reagent that is partof the hCG FLEX® kit product. The experimental reagents were then usedto replace the hCG conjugate in commercially approved DIMENSION® RxL hCGFLEX® kits. These experimental kits were run using the standard softwarefor the hCG Dimension method, using Level 4 calibrator, which contains529 mIU hCG. The results of the testing are shown in Table 1 below:

TABLE 1 Assay Response Change Response Compared to Quench CompoundConcentration (mAU) Control Sodium Azide 5 mM 475 13% 25 mM 528 26% 125mM 546 30% Sodium Cyanide 5 mM 429  2% 25 mM 371 −12%   125 mM 166−61%   Thiophenol 5 mM 546 30% 25 mM 430  2% 125 mM 337 −20%   Hydrazine5 mM 562 34% 25 mM 528 26% 125 mM 535 27% Mercaptoethylamine 5 mM 394 −6%   25 mM 420  0% 125 mM 439  4% Control No second quench 420  0%As can be seen, the largest increase in activity was achieved withhydrazine as the nucleophilic deactivation reagent. Thiophenol also gavean adequate response.

Example 2

Part A

A solution of 500 mg of anti-CKMB Fab′2 antibody was prepared at aconcentration of 8-10 mg/mL in 10 mM pH 7.0 phosphate buffer containing300 mM NaCl. To this solution, 5.014 mL of a 10 mg/mL SMCC solution inDMF (dimethylformamide) was added dropwise with stirring. The mixture isthen incubated for 30 minutes at 25° C., and then purified by passagethrough a SEPHADEX G-25® size exclusion column. The elution buffer wasthe same as the reaction buffer. The concentration of product was thendetermined using the OD 280 (assume the extinction coefficient is 1.4),and the concentration was adjusted to 2 mg/mL by addition of additionalreaction buffer.

Part B

A solution of 2700 mg of β-galactosidase in 1350 mL of 10 mM pH 7.0phosphate buffer containing 300 mM NaCl was prepared and brought to 25°C. with stirring. When the solution had reached this temperature thesolution of activated antibody from the first step above was added. Themixture was incubated at 25° C. for 3 hours and 10 minutes, and then thereaction was stopped by adding 32 mL of a 100 mM stock solution ofN-ethylmaleimide (NEM) in water.

Part C

Fifteen (15) minutes after addition of the NEM solution, 2 mL aliquotswere removed from the product mixture and 0.04 mL of a 10% stocksolution of TWEEN 20® was added to each aliquot. Sodium azide andhydrazine were added to two of the aliquots, and the third had noadditive. The concentration of sodium azide in the first was 125 mM andthe concentration of hydrazine in the second was 5 mM. The three sampleswere incubated for 18 hours at 20° C., and then purified by HPLC using aGF450XL® preparative column from Agilent Technologies Inc (Palo AltoCalif.). The product peak was eluted with a buffer containing 11.1 g/Lmonobasic sodium phosphate, 15.3 g/L dibasic sodium phosphate, and 0.2g/L sodium azide at pH 7.0. The peak that contained higher molecularweight material than β-galactosidase was collected, and other peaks werediscarded. Each of the three samples was adjusted to a concentrationsuch that the optical density of the product at 280 nm was 1 AU.

The mixtures were filtered through 0.2 micron filters and then testedover a period of 47 days, to determine their activity and stability.Half of the conjugates was stored at 4° C., and the other half wasstored at 25° C. On each test day, each experimental conjugate wasdiluted 16-fold with the conjugate diluent used for the DIMENSION® RxLcommercially available MMB conjugate reagent that is part of the MMBFLEX® kit product. These experimental reagents were then used to replacethe MMB conjugate in commercially approved DIMENSION® RxL MMB FLEX®kits. The experimental kits were tested using the standard software forthe MMB DIMENSION® RxL method with a calibrator that contains 133 ng/mLof CKMB. The results of the testing are shown in Table 2 below:

TABLE 2 Storage Response Response as Percent of Day 0 Quench ConditionTemperature on Day 0 Day 5 Day 12 Day 19 Day 47 NEM only (control)  4 C.301 97.5 95.8 93.8 88.1 5 mM Hydrazine*  4 C. 383 100.2 102.3 103.0 96.3150 mM Sodium Azide*  4 C. 410 96.8 97.1 95.3 92.1 NEM only (control) 25C. 301 94.1 92.2 86.1 87.7 5 mM Hydrazine* 25 C. 383 100.5 96.1 96.088.7 150 mM Sodium Azide* 25 C. 410 92.6 86.5 84.3 81.9 *The hydrazineand azide quench examples were first quenched with NEM

As discussed above, a desirable goal for conjugate stability is thatthere be less than a 5% change in response over one month for productstored at 4° C. The conjugate that was quenched with both NEM andhydrazine fulfilled this criterion while the conjugate quenched withboth NEM and azide did not. Accordingly, in some embodiments thenucleophilic reagent is substantially free of azide in that it containsless than about 0.1%, less than about 0.05%, less than about 0.01%, or0%, of azide.

Example 3

In this example, conjugates were prepared at commercial scale for assaysfor the detection of HCG, MMB, ferritin (FERR), and cyclosporin A (CSA),respectively, on the DIMENSION® RxL following the manufacturersdirections.

Part A

A solution of Fab′2 antibody (either 500 mg of anti-CKMB, 450 mg ofanti-HCG, or 200 mg of anti-FERR) or intact antibody (125 mg ofanti-CSA) was prepared at a concentration of 8-10 mg/mL in 10 mM pH 7.0phosphate buffer containing 300 mM NaCl. To this solution, 30-fold molarexcess of 10 mg/mL SMCC solution in DMF was added dropwise withstirring. The mixture was then incubated for 30 minutes at 25° C. andthen purified by passage through a SEPHADEX G-25® size exclusion column.The elution buffer was the same as the reaction buffer. Theconcentration of product was then determined using the OD 280 (assumingan extinction coefficient of 1.4), and the concentration was adjusted to2 mg/mL by addition of additional reaction buffer.

Part B

A solution of 2 mg/mL of β-galactosidase in 10 mM pH 7.0 phosphatebuffer containing 300 mM NaCl was prepared and brought to 25° C. withstirring. When the solution had reached this temperature, the solutionof the appropriate activated antibody from the first step above wasadded. The molar quantity of β-galactosidase was equal to that of theactivated antibody. The mixture was incubated at 25° C. for 3 hours and10 minutes, and then the reaction was stopped by adding a sufficientvolume of a 100 mM stock solution of N-ethylmaleimide (NEM) in water tobring the final NEM concentration to 1 mM.

Part C

Fifteen (15) minutes after addition of the NEM solution, the solutionwas divided into 2 equal aliquots. Hydrazine was added to one aliquot,and the other had no additive. The concentration of hydrazine was 5 mM.After incubating for 15 minutes at room temperature, the aliquots wereconcentrated using a filter concentration unit. The concentratedaliquots were then purified using a SEPHADEX S200® column. The productpeak was eluted with a buffer containing 11.1 g/L monobasic sodiumphosphate, 15.3 g/L dibasic sodium phosphate, and 0.2 g/L sodium azideat pH 7.0. The peak that contained higher molecular weight material thanα-galactosidase was collected, and other peaks were discarded. Each ofthe samples was adjusted to a concentration such that the opticaldensity of the product at 280 nm was 1 AU. The mixtures were filteredthrough 0.2 micron filters and then tested for specific activity.

The specific activity of a conjugate is dependent upon both itsenzymatic (β-Galactosidase) and immunologic (antibody binding) activity.For a conjugate with a given amount of enzymatic activity per molecule,the specific activity increases as the percent of Enzymatic ActivityBound to Active Antibody increases. Active antibody is defined asantibody capable of binding the antigen of interest (sbp member). Anincrease of a conjugate's specific activity allows less of the conjugateto be used per test on a clinical chemistry analyzer such as, forexample, the Dimension® RxL, when, e.g., a non-competitive format suchas a sandwich immunoassay is used.

For sandwich immunoassays such as the HCG, MMB, and FERR methods on theDIMENSION® RxL, the percentage of Enzymatic Activity Bound to ActiveAntibody is determined using the following procedure.

-   -   a. Prepare goat anti-mouse coated chrome particles (Pierce        product # 21354, MAGNABIND™ Goat anti-Mouse IgG Beads) according        the manufacturer's instructions.    -   b. Hydrate method-specific chrome tablets in diluent (specific        to each method) at 3 times the normal concentration (i.e., 3×        the concentration after hydration in a FLEX® well).    -   c. Prepare an antigen solution (method-specific) 10 to 100 times        (optimized for each method) the upper end of method's assay        range. Use 6% BSA for this solution.    -   d. Dilute each method-specific conjugate concentrate, in the        method's conjugate diluent, to the following dilutions. 1:20        (MMB) or 1:25 (FERR and HCG). Do this for both test and control        conjugate concentrates.    -   e. In a screw top plastic test tube, mix water, hydrated chrome,        antigen, and diluted conjugate at the ratios in the        immuno-reaction vessel (from the DIMENSION® method parameters        for each specific method).    -   f. In addition, in a screw top plastic test tube, set up the        same immuno-reaction mixture, except that 6% BSA is used in        place of the antigen solution.    -   g. Place the test tubes on a rocker and rock the tubes for 2        hours at room temperature.    -   h. Add 5 μL of the goat anti-mouse coated chrome reagent to each        tube and continue rocking for another 2 hours at room        temperature.    -   i. Remove the chrome using magnetic separation and test the        supernatants as samples using the appropriate DIMENSION® method,        in triplicate. Collect filter data and use the short reads for        each method.    -   j. The total enzymatic activity is defined as the mean result        from the tube with no antigen.    -   k. The enzymatic activity that did not bind chrome is defined as        the mean result from the tube with antigen.    -   l. The total enzymatic activity that is bound to active antibody        is the difference between the total and non-binding enzymatic        activity.

The results of the testing are summarized in Table 3 below:

TABLE 3 Accep- Control Test Yield MMB Control Test tance DilutionDilution Increase Lot # % Bound % Bound Criterion Factor Factor (%)190141 96.8 97.2 Test ≧ 8.4 12.1 144 Control 193429 94.9 95.5 Test ≧ 9.516.7 176 Control 195810 96.1 96.7 Test ≧ 9.0 15.9 177 Control Accep-Control Test Yield HCG Control Test tance Dilution Dilution Increase Lot# % Bound % Bound Criterion Factor Factor (%) 190197 52 56 Test ≧ 12.520 160 Control 193428 54 66 Test ≧ 8 20 250 Control 195811 61 76 Test ≧12 25 208 Control Accep- Control Test Yield FERR Control Test tanceDilution Dilution Increase Lot # % Bound % Bound Criterion Factor Factor(%) 191009 61 64 Test ≧ 12 16 133 Control

The CSA method uses ACMIA immunoassay technology on the DIMENSION® RxL.This method employs competition between antigen (cyclosporine) on chromeparticles and antigen in patient samples to determine the amount ofcyclosporine in the patient samples. Conjugate that binds the antigen onchrome particles is removed by magnetic separation. The enzymaticactivity from conjugate remaining in the supernatant is measured and isdirectly proportional to the amount of antigen in the patient sample.When an ACMIA assay format is employed, the enzymatic activity observedwhen testing a sample containing no antigen is indicative of the amountof enzymatic activity that is not bound to active antibody (i.e., cannotbind antigen on chrome). The enzymatic activity observed when no chromeis present is indicative of the total amount of enzymatic activity inthe conjugate. These values can be used to estimate the percent ofenzymatic activity bound to active antibody.

Results from such a study are shown in Table 4.

TABLE 4 0 ng/mL Sample No Chrome Lot # mAU mAU % Not Bound AcceptanceCriterion 190104 (Control - Dilution 151.9 1224.6 12 Test ≧ ControlFactor 190) 190104A (Test - Dilution 109.0 1165.5 9 Factor 190) 190104(Control - Dilution 365.8 786.7 46 Test ≧ Control Factor 300) 190104A(Test - Dilution 280.3 760.3 37 Factor 300) 190486(Control - Dilution185 1728 11 Test ≧ Control Factor 190) 190486A (Test - Dilution 134 17018 Factor 190) 190486 (Control - Dilution 245 1233 20 Test ≧ ControlFactor 300) 190486A (Test - Dilution 174 1231 14 Factor 300)194016(Control - Dilution 161 1409 11 Test ≧ Control Factor 190) 194016A(Test - Dilution 144 1479 10 Factor 190) 194016 (Control - Dilution 901347 7 Test ≧ Control Factor 275) 194016A (Test - Dilution 62 1309 5Factor 275)

It is desirable for conjugates to have high specific activities in theimmunoassays for which they are manufactured. The conjugates that werequenched with both NEM and hydrazine (di-deactivated) had higherspecific activity than conjugates that were only quenched with NEM.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims. Furthermore, the foregoing description,for purposes of explanation, used specific nomenclature to provide athorough understanding of the invention. However, it will be apparent toone skilled in the art that the specific details are not required inorder to practice the invention. Thus, the foregoing descriptions ofspecific embodiments of the present invention are presented for purposesof illustration and description; they are not intended to be exhaustiveor to limit the invention to the precise forms disclosed. Manymodifications and variations are possible in view of the aboveteachings. The embodiments were chosen and described in order to explainthe principles of the invention and its practical applications and tothereby enable others skilled in the art to utilize the invention.

1. A conjugate comprising a first polypeptide and a second polypeptidelinked together by sulfhydryl groups bound to sulfhydryl receptorswherein sulfhydryl receptors not bound to sulfhydryl groups are in theform of a product of said sulfhydryl receptor and a sulfhydryl receptordeactivation reagent wherein said sulfhydryl receptor deactivationreagent has the formula:R²HN—(R₁ )_(n)—NHR² wherein n is 0 or 1, R¹ is alkylene of 1 to about 5carbon atoms and R² is independently H, alkyl or substituted alkyl.
 2. Aconjugate according to claim 1 wherein the sulfhydryl receptordeactivation reagent is substantially non-reactive with disulfidelinkages when said conjugate comprises disulfide linkages
 3. A conjugateaccording to claim 1 wherein said sulfhydryl receptor deactivationreagent is hydrazine.
 4. A conjugate according to claim 1 wherein saidpolypeptides are independently selected from the group consisting ofantibodies and enzymes.
 5. A conjugate according to claim 1 wherein saidsulfhydryl receptor comprises a 1,2-di-carbonyl substituted olefin, asulfone substituted olefin, or a carbonyl substituted, halogensubstituted carbon atom.
 6. A conjugate according to claim 5 whereinsaid sulfhydryl receptor comprises a 1,2-di-carbonyl substituted olefinthat is a maleimide.
 7. A conjugate according to claim 5 wherein saidsulfhydryl receptor comprises a carbonyl substituted, halogensubstituted carbon atom that is a halo-acetyl moiety.
 8. A conjugateaccording to claim 5 wherein said sulfhydryl receptor comprises asulfone-substituted olefin wherein the olefin bond is conjugated withthe sulfone.
 9. A conjugate according to claim 1 wherein sulfhydrylgroups not bound to sulfhydryl receptors are deactivated.
 10. In amethod for conjugating a first compound to a second compound whereinsaid conjugation involves an electrophilic moiety and said methodcomprises reacting said first compound with said second compound to forma conjugate, the improvement comprising adding a nucleophilic reagent tosaid conjugate wherein said nucleophilic reagent has the formulaR²HN—(R₁)_(n)—NHR², wherein n is 0 or 1, R₁ is an alkylene of 1 to about5 carbon atoms and R² is independently H, alkyl or substituted alkyl andforms a product upon reaction with unreacted electrophilic moieties ofsaid conjugate wherein said product is the conjugate according toclaim
 1. 11. A method according to claim 10 wherein said electrophilicmoiety comprises a sulfhydryl receptor.
 12. A method according to claim10 wherein said first compound and said second compound areindependently selected from the group consisting of enzymes and ligandbinders.
 13. A method according to claim 12 wherein said ligand bindersare polypeptides or polynucleotides.
 14. A method according to claim 10wherein said first compound comprises one or more sulfhydryl groups andsaid second compound comprises more than one sulfhydryl receptor.
 15. Amethod according to claim 10 wherein said sulfhydryl receptor comprisesa carbonyl substituted olefin, a sulfone substituted olefin, or acarbonyl substituted, halogen substituted carbon atom.
 16. A methodaccording to claim 10 wherein, when said conjugate comprises disulfidebonds, said nucleophilic reagent is substantially non-reactive withdisulfide bonds.
 17. A method for conjugating a first polypeptide to asecond polypeptide wherein said conjugation involves a sulfhydrylreceptor, said method comprising: (a) combining said first polypeptidewith said second polypeptide under conditions for said first polypeptideand said second polypeptide to react to form a conjugate involving saidsulfhydryl receptor and (b) adding a sulfhydryl receptor deactivationreagent having the formula R²HN—(R₁)_(n)—NHR². wherein n is 0 or 1, R₁is an alkylene of 1 to about 5 carbon atoms and R² is independently H,alkyl or substituted alkyl, to said conjugate wherein said sulfhydrylreceptor deactivation reagent forms the conjugate according to claim 1.18. A method according to claim 17 wherein said first polypeptide andsaid second polypeptide are independently selected from the groupconsisting of antibodies and enzymes.
 19. A method according to claim 18wherein said first polypeptide is an enzyme and said second polypeptideis an antibody.
 20. A method according to claim 17 wherein said firstpolypeptide comprises one or more sulfhydryl groups and said secondpolypeptide comprises more than one sulfhydryl receptor.
 21. A methodaccording to claim 17 wherein said sulfhydryl receptor comprises acarbonyl substituted olefin, a sulfone substituted olefin, or a carbonylsubstituted, halogen substituted carbon atom.
 22. A method according toclaim 21 wherein said sulfhydryl receptor comprises a carbonylsubstituted olefin that is a 1,2-di-carbonyl substituted olefin.
 23. Amethod according to claim 22 wherein said 1,2-di-carbonyl substitutedolefin is a maleimide.